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Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose-response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.
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Proteínas , Proteínas/química , Espectrometria de Massas , Pegadas de Proteínas/métodos , Conformação Proteica , Aminoácidos/química , Dicroísmo CircularRESUMO
Polycythemia vera is a Philadelphia chromosome-negative myeloproliferative neoplasm characterized by the clonal proliferation of hematopoietic cells, leading to the overproduction of erythrocytes and the elaboration of inflammatory cytokines. Management is aimed at reducing the risk of thromboembolic events, alleviating the symptom burden, decreasing splenomegaly, and potentially mitigating the risk of disease progression. Existing treatment options include therapeutic phlebotomy and cytoreductive agents including hydroxyurea, pegylated recombinant interferon alpha 2a, ropegylated recombinant interferon alpha 2b, and ruxolitinib. We review risk factors for both thrombotic events and disease progression in patients with polycythemia vera. We discuss existing and novel therapeutic approaches to mitigate the risk of disease-related complications and progression.
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Policitemia Vera , Humanos , Policitemia Vera/tratamento farmacológico , Policitemia Vera/genética , Objetivos , Eritrócitos , Fatores de Risco , Interferon alfa-2 , Progressão da DoençaRESUMO
In the published publication [...].
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BACKGROUND: Asthma is a leading cause of children's hospitalizations, emergency department visits, and missed school days. Our school-based asthma intervention has reduced asthma exacerbations for children experiencing health disparities in the Denver Metropolitan Area, due partly to addressing care coordination for asthma and social determinants of health (SDOH), such as access to healthcare and medications. Limited dissemination of school-based asthma programs has occurred in other metropolitan and rural areas of Colorado. We formed and engaged community advisory boards in socioeconomically diverse regions of Colorado to develop two implementation strategy packages for delivering our school-based asthma intervention - now termed "Better Asthma Control for Kids (BACK)" - with tailoring to regional priorities, needs and resources. METHODS: In this proposed type 2 hybrid implementation-effectiveness trial, where the primary goal is equitable reach to families to reduce asthma disparities, we will compare two different packages of implementation strategies to deliver BACK across four Colorado regions. The two implementation packages to be compared are: 1) standard set of implementation strategies including Tailor and Adapt to context, Facilitation and Training termed, BACK-Standard (BACK-S); 2) BACK-S plus an enhanced implementation strategy, that incorporates network weaving with community partners and consumer engagement with school families, termed BACK-Enhanced (BACK-E). Our evaluation will be guided by the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework, including its Pragmatic Robust Implementation Sustainability Model (PRISM) determinants of implementation outcomes. Our central hypothesis is that our BACK-E implementation strategy will have significantly greater reach to eligible children/families than BACK-S (primary outcome) and that both BACK-E and BACK-S groups will have significantly reduced asthma exacerbation rates ("attacks") and improved asthma control as compared to usual care. DISCUSSION: We expect both the BACK-S and BACK-E strategy packages will accelerate dissemination of our BACK program across the state - the comparative impact of BACK-S vs. BACK-E on reach and other RE-AIM outcomes may inform strategy selection for scaling BACK and other effective school-based programs to address chronic illness disparities. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT06003569, registered on August 22, 2023, https://classic. CLINICALTRIALS: gov/ct2/show/NCT06003569 .
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Asma , Serviços de Saúde Escolar , Humanos , Asma/terapia , Asma/prevenção & controle , Criança , Colorado , Serviços de Saúde Escolar/organização & administração , Adolescente , Populações Vulneráveis , Ciência da Implementação , FemininoRESUMO
BACKGROUND: Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. METHODS: We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca2+ kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tvHeLa). RESULTS: Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca2+ concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to ß-adrenergic stimulation. CONCLUSIONS: Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca2+ kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.
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Cardiomiopatia Dilatada , Ataxia Cerebelar , Células-Tronco Pluripotentes Induzidas , Maleatos , Erros Inatos do Metabolismo , Humanos , Trifosfato de Adenosina/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Células HeLa , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , RespiraçãoRESUMO
Microglia help limit the progression of Alzheimer's disease (AD) by constraining amyloid-ß (Aß) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. Here, we find that LILRB4 is highly expressed in the microglia of patients with AD. Using mice that accumulate Aß and carry a transgene encompassing a portion of the LILR region that includes LILRB4, we corroborated abundant LILRB4 expression in microglia wrapping around Aß plaques. Systemic treatment of these mice with an anti-human LILRB4 monoclonal antibody (mAb) reduced Aß load, mitigated some Aß-related behavioral abnormalities, enhanced microglia activity, and attenuated expression of interferon-induced genes. In vitro binding experiments established that human LILRB4 binds both human and mouse ApoE and that anti-human LILRB4 mAb blocks such interaction. In silico modeling, biochemical, and mutagenesis analyses identified a loop between the two extracellular Ig domains of LILRB4 required for interaction with mouse ApoE and further indicated that anti-LILRB4 mAb may block LILRB4-mApoE by directly binding this loop. Thus, targeting LILRB4 may be a potential therapeutic avenue for AD.
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Doença de Alzheimer , Microglia , Humanos , Camundongos , Animais , Microglia/metabolismo , Anticorpos/metabolismo , Receptores de Superfície Celular/metabolismo , Amiloide/metabolismo , Modelos Animais de Doenças , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E , Leucócitos/metabolismo , Camundongos Transgênicos , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Context: School-based asthma programs (SBAPs) have improved health and educational disparities among youth with asthma. Design: To support scaling out effective SBAPs, our school partners identified a need for online implementation guides that are "always available," to meet the needs of school nurses' demanding schedules. School nurses play a key role in the adoption and implementation of SBAPs, so it is important to ensure the implementation guide would be highly usable and acceptable to them. Objective: Accordingly, our research team collaborated with human-centered design experts to identify the "user journeys" of school nurses and co-created our online implementation guide as a public-facing website with input from local and national school nurse partners. Main Results: In this perspectives article, our school nurse implementation partners and human-centered design experts reflect on challenges overcome in this process of developing a tailored implementation guide to school nurses and offer lessons from the field to others seeking to co-create implementation guides with community partners.